Mixed cell diagnostic systems

ABSTRACT

The present invention generally relates to the field of diagnostic microbiology, and, more particularly, to compositions and methods for detecting and differentiating one or more viruses or other intracellular parasites present in a specimen. The present invention also provides compositions and methods to evaluate the susceptibility of a organisms to antimicrobial agents.

This is a continuation of application Ser. No. 09/661,849, filed on Sep.14, 2000, now U.S. Pat. No. 6,376,172, which is a divisional ofapplication Ser. No. 09/066,072, filed on Apr. 24, 1998, and issued asU.S. Pat. No. 6,168,915 on Jan. 2, 2001.

FIELD OF THE INVENTION

The present invention generally relates to the field of diagnosticmicrobiology, and more particularly, to compositions and methods fordetecting and differentiating one or more viruses or other intracellularparasites present in a specimen. The present invention also providescompositions and methods to evaluate the susceptibility of a organismsto antimicrobial agents.

BACKGROUND OF THE INVENTION

Despite recent advances in methods for the detection of viruses usingmolecular methods, the detection and identification of these organismsin cell culture remains the “hold standard” by which most viral diseasesare definitively diagnosed and the method against which newer methodsare compared (See e.g., Wiedbrauk and Johnston, Manual of ClinicalVirology, Raven Press, Inc., New York, N.Y. [1993], pp. 1-17). Cellcultures are also used for the detection and identification of otherintracellular parasites, especially obligate intracellular parasitessuch as Chlamydia and Rickettsia.

There are two general types of cell culture methods used for virusidentification. The first method uses identification of virus-inducedcytopathic effect (CPE) as an endpoint for virus detection. The secondmethod utilizes molecular methods to identify the presence of virusbefore CPE is evident in the infected cultures. Both methods utilizecell cultures, which may present problems for small laboratories withlimited expertise in cell culturing methods, space, funding, equipment,and supplies. Depending upon the cells used, cell cultures can bedifficult to maintain and often require the efforts of skilledlaboratorians. In addition, cell cultures require equipment such as cellculture hoods, inverted microscopes (for observation of cells),incubators with CO₂ lines, and other equipment not readily available inmany laboratories.

CPE-Based Tests

CPE-based tests often require long incubation times, as virus-inducedCPE only becomes evident after multiple rounds of viral replication andspread of virus to neighboring cells (i.e., the cells are “permissive”for viral infection). Virus spread results in the destruction of thecells surrounding the cell originally infected. CPE-based tests havebeen traditionally conducted in tubes or flasks containing a single celltype that is adhered or anchored to the sides and/or bottom of the tubeor flask. As the virus must infect a cell, replicate, and spread toneighboring cells in which the process is repeated, results can bedelayed for at least 28 days. Indeed, results are often not availablefor 7-28 days after inoculation of the cell culture with the virussuspension (See e.g., Leland, Clinical Virology, W. B. Saunders,Philadelphia [1996], pp. 60-65). The time necessary to establish visibleCPE is dependent upon the rate of viral replication, which can varyamong cell types and viruses. Thus, the amount of time needed to detectvirus in a sample can greatly vary.

Pre-CPE Tests

In contrast to CPE-based tests, pre-CPE tests require only entry of thevirus into a susceptible host cell and detectable expression of at leastone early virus-specific antigen or nucleic acid. Detection of thevirus-specific analyte or other indicator is accomplished by a number ofmethods (e.g., labeled antibodies, the polymerase chain reaction [PCR],or the use of other reporters, such as the ELVIS™ system). Expression ofearly viral genes has been shown to be very rapid in many virus-hostcell systems in vitro. Thus, use of pre-CPE based virus testssignificantly reduces the time required to detect and identify virusesin clinical specimens.

Pre-CPE detection of virus is often accomplished by using monolayers ofadherent cells grown on 12 mm round coverslips contained in 1 dram shellvials (i.e., the “shell vial” method or technique). The shell vialtechnique uses centrifugation of the specimen to force viralintroduction into cells and enhance viral isolation. These vials areprepared by dispensing cells into sterile shell vials containingcoverslips. The vial are incubated in an upright position until thecells form a monolayer on the coverslip. For shell vial inoculation, theculture medium is decanted from the vial, processed sample (i.e., theclinical specimen) is added to the cell monolayer, and the vial iscentrifuged at low speed, often for one hour. After centrifugation,fresh culture medium is added to each vial. The vials are then incubatedfor the desired period of time. At the end of the incubation period, thecoverslips are stained using an antigen detection method (e.g.,immunofluorescence) or the cells are evaluated via molecular diagnostictechniques.

In addition to viruses, shell vials are also commonly used for thedetection and identification of Chlamydia, as other methods availablefor the detection and identification of these organisms are quitecumbersome, as well as time and reagent-consuming (See e.g., Wiedbraukand Johnston, supra, pp. 64-76).

The major advantage of these pre-CPE testing methods is that rapid testresults are often possible. One major disadvantage to pre-CPE testing ofshell vial cultures is that this type of test is feasible andcost-effective only if one or a few viral agents are sought foridentification, and if a high proportion of specimens are likely to bepositive (See e.g., Schmidt and Enunons, “General Principles ofLaboratory Diagnostic Methods for Viral, Rickettsial, and ChlamydialInfections,” in Schmidt and Emmons (eds.), Diagnostic Procedures forViral, Rickettsial and Chlamydial Infections, American Public HealthAssociation, Washington, D.C., [1989], at p. 4).

Clinical Specimens

For example, the presence of skin vesicles in the genital area of apatient is highly suspicious for infection by herpes simplex virus(HSV). Typically, the physician will obtain a specimen from the affectedregion (i.e., a vesicle) and order a CPE or a pre-CPE virus test on asingle, HSV-susceptible cell line. These cell lines are often suppliedeither in tubes, shell vials, or multi-well plates (e.g, microtiterplates). After inoculation of the cell line and an appropriateincubation time, confirmation of the presence of HSV in the sample canbe accomplished using one or more of the many analytical methods (e.g.,immunofluorescence, immunoperoxidase, nucleic acid probes, or substratesfor virus-induced reporter genes).

For detection of cytomegalovirus (CMV), shell vials containing cellsfrom a single cell line (e.g., human fibroblast cell lines, such as lung[MRC-5 cells] or foreskin [HFF] cells) are often used. The cells aregrown to confluency on the coverslip within the vial, the sample isadded to the vial, the vial is incubated for 24-48 hours or longer, andan immunofluorescent method is used to detect expression of CMV earlyantigen.

Accurate differential diagnosis is significantly more difficult in virusdiseases due to respiratory, gastrointestinal, genital, or parenteralroutes of transmission because many pathogenic viruses are capable ofeliciting similar symptoms or the infection is sub-clinical (i.e., thesigns and symptoms are not readily apparent).

Of the respiratory viruses, rhinoviruses and corona viruses areresponsible for a large proportion of upper respiratory infections. Oncethese viruses reach the upper respiratory mucosa, they attach to andinfect epithelial cells. Typically, these infections last only a fewdays and self-resolve. Other respiratory viruses, such as theinfluenzas, parainfluenzas, respiratory syncytial virus (RSV), andvarious adenoviruses attach to and infect ciliated, columnar epithelialcells. The virus-infected cells lyse, resulting in the release ofenzymes and activate complement, resulting in a local mononuclearinflammatory response. Normal airway clearance mechanisms fail becauseof the failure of the epithelial cells to function normally. These cellsmay also slough off. Cell debris from dead and dying cells oftenobstructs airways, and the host becomes very susceptible to secondarybacterial infection and/or superinfection. All of these viruses mayprogress to lower respiratory involvement and pneumonia. Afterreplication in the respiratory epithelial cells, adenovirus may travelvia the blood to the lymphoid tissues in all areas of the body, causingsystemic infection or disease.

Standard clinical virology practice is to inoculate multiple tubes ofcell cultures with the specimen (e.g., throat swab, nasopharyngeal swab,or sputum specimen) as the tropism of each type of virus for specificcell types is often very narrow (i.e., only one type of virus may growoptimally on a single cell type). This narrow tropism of virus for alimited number of cell types creates at least two major practicalproblems for both CPE and pre-CPE virus testing.

First, primary monkey kidney cells are currently the cell line of choicefor isolation of influenza viruses. The manufacture of these cellsrequires the quarantine of source animals for long periods prior tosacrifice and cell culture preparation. This quarantine period is usedto monitor the animals for good health and allows time to test theanimals for infection by endogenous simian viruses such as foamy virus,SV5, and SV40. The quarantine period also greatly reduces, but does noteliminate, the possibility that the monkeys are infected with Monkey BVirus, a herpesvirus that is highly fatal to humans. In addition, thereare other problems related to the use of monkeys for the production ofprimary cell cultures, including the reduction in the stock of suitableanimals due to importation concerns and monkey populationconsiderations.

Second, additional continuous cell lines are required in order to detectrespiratory viruses other than influenza virus. Thus, multiple celllines are used in order to diagnose the viral infection/disease of eachpatient. The need for multiple units of individual cell lines iscompounded in methods using pre-CPE tests for detection andidentification of respiratory viruses. Pre-CPE testing for respiratoryviruses requires the expenditure of significant labor in handlingcoverslips, the added expense of molecular reagents used with multiplecell lines for both positive and negative specimens, and the significantlabor associated with microscopically reading each of the multiple celllines inoculated in the panel of cell lines.

However, despite these drawbacks, shell vial technology using singlecell types in multiple units (tubes, shell vials, etc.), is stillcurrently used to detect respiratory viruses, as it is a proven method.For example, detection of RSV in 16 hours using shell vials containingonly HEp-2 cells yielded more positives than antigen detection methodsapplied directly to the clinical specimen, and as many positives asconventional cell cultures (Smith et. al., J. Clin. Microbiol.,29:463-465 [1991]). Isolation of other respiratory viruses has also beenpossible with shell vial cultures containing a monolayer of a singlecell type. For example, using vials of primary monkey kidney cells andA549 cells incubated for 40 hours, 83% of adenoviruses, 94% of influenzaB, and 80% of parainfluenza virus types 1, 2, and 3 were identified(Rabalais et al., J. Clin. Microbiol., 30:1505-1508 [1992]). In anotherreport, 50% of adenoviruses, 94% of influenza A viruses, 100% ofinfluenza B viruses, and 100% of parainfluenza viruses, in shell vialsof primary rhesus monkey kidney cells, and 92% of RSV in shell vials ofHEp-2 cells incubated for 24 days (See e.g., Olsen et al., J. Clin.Microbiol., 31:422-425 [1993]; and Leland, Clinical Virology, W. B.Saunders Company, Philadelphia, Pa. [1996], at p. 85-86).

Although these methods provide relatively rapid results (i.e., asopposed to the long incubation periods often necessary for CPE tests),there remains a need in clinical and reference virology laboratories forcell culture methods and compositions for the reliable detection andidentification of viruses in a single, easy-to-manipulate unit thatprovides rapid detection and identification in a cost-effective manner,while also providing the sensitivity of a diagnostic assay system.

SUMMARY OF THE INVENTION

The present invention generally relates to the field of diagnosticmicrobiology, and more particularly, to compositions and methods fordetecting and differentiating one or more viruses or other intracellularparasites present in a specimen. The present invention also providescompositions and methods to evaluate the susceptibility of a organismsto antimicrobial agents.

In particular, the present invention provides methods and compositionssuitable for the detection of viruses using CPE-based and pre-CPEmethods. The preferred embodiments encompass mixed cell cultures whichcontain at least two different cell types. In some preferredembodiments, the mixed cell cultures contain two different cell types,while in other embodiments, the mixed cell cultures contain three ormore different cell types. Thus, it is intended that the presentinvention encompass compositions in which at least two cell types aremixed together in one container (e.g., flask, tube, shell vial, or anyother container suitable for the growth of cells). Importantly, eachcell type within these mixed cell cultures retains its susceptibility toviruses and other intracellular parasites as if it was in a single cellculture (i.e., a cell culture that contains only one cell type, as knownin the art). In addition, the mixed cell cultures of the presentinvention remain viable for as long as required for their use indiagnostic assays. In particularly preferred embodiments, the cell typesincluded within mixed cell cultures are present in approximately thesame ratio (i.e., for a two cell type mixed, there is a 50:50 ratio ofcell types). However, it is not intended that the present invention belimited to any particular ratio of cell types in mixed culture, asvarious detection systems may be optimized using different ratios. Forexample, in some circumstances, a two cell mixture of 45:55, 40:60, oreven 35:75, may be more suited than a 50:50 ratio.

The present invention also provides methods and compositions suitablefor the detection and identification of non-viral obligate intracellularand intracellular parasites, such as members of the Chlamydiales andRicketsiales.

The present invention also contemplates compositions comprising a cellculture suitable for the detection of intracellular parasites, whereinthe cell culture comprises at least two cell types susceptible toinfection by at least one intracellular parasite. In some preferredembodiments of the composition, the cell types comprise a first celltype and a second cell type. In some embodiments, the first cell typeconsists of buffalo green monkey kidney cells and the second cell typeconsists of mink lung cells. In other embodiments, the first cell typeconsists of mink lung cells and the second cell type consists of humanmucoepidermoid cells. In yet other embodiments, the first cell typeconsists of human lung carcinoma cells and the second cell type consistsof human mucoepidermoid cells. In still other embodiments, the firstcell type consists of buffalo green monkey kidney cells and the secondcell type consists of human embryonic lung cells. In furtherembodiments, the cell type consists of human epidermoid laryngealcarcinoma cells and the second cell type consists of McCoy cells. Inadditional embodiments, the first cell type consists of mink lung cellsand the second cell type consists of human diploid lung cells.

In some preferred embodiments, the cell types of the composition aresusceptible to respiratory viruses, including but not limited toinfluenza viruses of any type (e.g., Influenza A, Influenza B, andInfluenza C) and/or strain, RSV, cytomegalovirus, parainfluenza viruses,respiratory syncytial virus, rhinoviruses, coronoviruses, andadenoviruses. In yet other embodiments, the cell types of thecomposition are susceptible to enteroviruses, including but not limitedto any type and/or strain of echovirus, poliovirus, and Coxsackie virus(e.g., Coxsackie A and B viruses), and numbered EV strains. In additionto enteroviruses, it is contemplated that the present inventionencompass cell types that are susceptible to picornaviruses such asHepatitis A.

The present invention also provides methods for the detection andidentification of intracellular parasites in a sample, comprising thesteps of providing a sample suspected of containing one or moreintracellular parasites; and a mixed cell culture comprising at leasttwo cell types; inoculating the mixed cell culture with the sample toproduce an inoculated culture; and observing the inoculated culture forthe presence of the one or more intracellular parasites.

In some embodiments of the method, the intracellular parasite is avirus. In some particularly preferred embodiments, the virus is selectedfrom the group consisting of cytomegalovirus, influenza viruses,parainfluenza viruses, respiratory syncytial virus, rhinoviruses,coronoviruses, and adenoviruses. In yet other embodiments of themethods, the virus is an enterovirus. In other particularly preferredembodiments, the enterovirus is selected from the group consisting ofpoliovirus, Coxsackie viruses and echoviruses (e.g., Coxsackie A and Bviruses), and numbered EV strains. In addition to enteroviruses, it iscontemplated that the present invention encompass cell types that aresusceptible to picornaviruses such as Hepatitis A. In still otherpreferred embodiments, the virus is a herpes virus. In otherparticularly preferred embodiments, the herpes virus is selected fromthe group consisting of Herpes Simplex Type 1, Herpes Simplex Type 2,Cytomegalovirus, Varicella-Zoster virus, Epstein-Barr virus, HumanHerpes Virus 6, Human Herpes Virus 7, and Human Herpes Virus 8. In yetother preferred embodiments, the intracellular parasite is a member ofthe genus Chlamydia. In still other particularly preferred embodiments,the intracellular parasite is C. trachomatis.

In some preferred embodiments of the methods, the cell types comprise afirst cell type and a second cell type. In some preferred embodiments,the first cell type is a mink lung cell, and the second cell type is ahuman mucoepidermoid cell. In other preferred embodiments, the firstcell type is a buffalo green monkey kidney cell and the second cell typeis a human mucoepidermoid cell. In yet another alternative embodiment,the first cell type is a genetically engineered baby hamster kidney celland the second cell type is a mink lung cell. In still otherembodiments, the first cell type is a first genetically engineered celltype and the second cell type is a second genetically engineered celltype.

It is contemplated that the methods of the present invention will beused in conjunction with controls of known positivity and negativity forthe virus(es) and/or other intracellular organism of interest.

The present invention also provides methods for the detection andidentification of intracellular parasites in a sample, comprising thesteps of providing: a sample suspected of containing one or moreintracellular parasites; and a mixed cell culture comprising a firstcell type and a second cell type; inoculating the mixed cell culturewith the sample to produce an inoculated culture; and observing theinoculated culture for the presence of the one or more intracellularparasites.

In some particularly preferred embodiments, the intracellular parasiteis a virus. In some particularly preferred embodiments, the virus isselected from the group consisting of cytomegalovirus, influenzaviruses, parainfluenza viruses, respiratory syncytial virus,rhinoviruses, coronoviruses, and adenoviruses. In yet other embodimentsof the methods, the virus is an enterovirus. In other particularlypreferred embodiments, the enterovirus is selected from the groupconsisting of poliovirus, Coxsackie viruses and echoviruses (e.g.,Coxsackie A and B viruses), and numbered EV strains. In addition toenteroviruses, it is contemplated that the present invention encompasscell types that are susceptible to picornaviruses such as Hepatitis A.In still other preferred embodiments, the virus is a herpes virus. Inother particularly preferred embodiments, the herpes virus is selectedfrom the group consisting of Herpes Simplex Type 1, Herpes Simplex Type2, Cytomegalovirus, Varicella-Zoster virus, Epstein-Barr virus, HumanHerpes Virus 6, Human Herpes Virus 7, and Human Herpes Virus 8. In yetother preferred embodiments, the intracellular parasite is a member ofthe genus Chlamydia. In still other particularly preferred embodiments,the intracellular parasite is C. trachomatis.

In some preferred embodiments of the methods, the cell types comprise afirst cell type and a second cell type. In some preferred embodiments,the first cell type is a mink lung cell, and the second cell type is ahuman mucoepidermoid cell. In other preferred embodiments, the firstcell type is a buffalo green monkey kidney cell and the second cell typeis a human mucoepidermoid cell. In yet another alternative embodiment,the first cell type is a genetically engineered baby hamster kidney celland the second cell type is a mink lung cell. In still otherembodiments, the first cell type is a first genetically engineered celltype and the second cell type is a second genetically engineered celltype.

It is contemplated that the methods of the present invention will beused in conjunction with controls of known positivity and negativity forthe virus(es) and/or other intracellular organism of interest.

The present invention further provides methods for the detection ofinfluenza virus, comprising the steps of providing a sample suspected ofcontaining influenza virus, and mink lung cells; inoculating the minklung cells with the sample; and detecting the presence of the influenzawithin the mink lung cells. In particularly preferred embodiments, themink lung cells are Mv1Lu cells. In alternative embodiments, theinfluenza virus is selected from the group consisting of Influenza A,Influenza B, and Influenza C.

It is contemplated that the methods of the present invention will beused in conjunction with controls of known positivity and negativity forthe virus(es) and/or other intracellular organism of interest.

In one embodiment, the present invention provides methods for thedetection of infectious virus in a specimen comprising the steps of: a)providing a specimen suspected of containing a virus, a cell linepermissive for infection by the virus, and a genetically engineered cellline containing an oligonucleotide having a sequence comprising apromoter sequence derived from the virus, wherein the promoter sequenceis operably linked to a reporter gene, and wherein the expression of thereporter gene is dependent upon and quantitatively proportional to thepresence of the virus; b) mixing together the permissive cell line andthe genetically engineered cell line to create a mixed cell culture; c)inoculating the mixed cell culture with the specimen under conditionswhich permit the infection of the mixed cell culture by the virus; andd) detecting the expression of the reporter gene and thereby detectingthe presence of virus in the specimen. In one preferred embodiment, themixed cell culture is a mixture consisting of 80-99% of the permissivecell line and 1-20% of the genetically engineered cell line. In otherpreferred embodiments, the mixed cell culture is a mixture consisting ofequal proportions of the cell types used in the mixture.

In one embodiment of the method, the genetically engineered cell linecontains an oligonucleotide having a sequence comprising a herpesvirusinducible promoter operably linked to a reporter gene selected from thegroup comprising the Escherichia coli lacZ gene and a luciferase gene.In one preferred embodiment of the method, the genetically engineeredcell line is BHKICP10LacZ. In an alternative preferred embodiment, thegenetically engineered cell line is BHKICP6LacZ. However, it is notintended that the reporter gene be limited to the lacZ and luciferasegenes. Indeed, it is contemplated that any suitable reporter gene knownto those in the art will be useful in the method of the presentinvention.

It is also contemplated that various permissive cell lines will beuseful in the method of the present invention. In one embodiment, thepermissive cell line is permissive for infection with herpesvirus. In aparticularly preferred embodiment, the permissive cell line is MRC-5.

It is contemplated that the method of the present invention will be usedin conjunction with controls of known positivity and negativity for thevirus(es) of interest. Thus, for mixed cultures in which geneticallyengineered cell lines are used, it is contemplated that the pattern ofreporter gene expression present in a test sample (e.g., from a clinicalspecimen) will be compared to the patterns of reporter gene expressionin control samples known to be positive and/or negative for thevirus(es) of interest. It is also contemplated that effects unrelated tothe expression of the reporter gene will be detectable, including butnot limited to CPE. These effects, alone and in combination with thereporter gene expression may be used to detect the presence of viralinfection.

The present invention also provides methods for the typing of infectiousherpesvirus in specimens, comprising the steps of: a) providing aspecimen suspected of containing one or more members of the herpesvirusfamily, a cell line permissive for infection by one or more members ofthe herpesvirus family, a genetically engineered cell line containing anoligonucleotide having a sequence comprising a promoter sequence derivedfrom a member of the herpesvirus family wherein the promoter sequence isoperably linked to a reporter gene, and the expression of the reportergene is dependent upon and quantitatively proportional to the presenceof herpesvirus and wherein the expression of the reporter gene varies ina distinguishable manner as a result of the presence of differentmembers of the herpesvirus family; b) mixing together the permissivecell line and the genetically engineered cell line to create a mixedcell culture; c) inoculating this mixed cell culture with the specimenunder conditions which permit the infection of the mixed cell culture bymembers of the herpesvirus family, wherein the infection results in adistinguishable pattern of expression by the reporter gene; d) detectingthe expression of the reporter gene and thereby detecting the presenceof one or more members of the herpesvirus family in the specimen; and e)identifying the presence of a specific member of the herpesvirus familybased upon the resulting distinguishable pattern. It is contemplatedthat this pattern of expression will be observable by various assistedand non-assisted methods, including visual observation by eye,spectrophotometric observation, etc. It is not intended that thedetection of distinguishable pattern(s) be limited to any particularmethod of detection.

In a preferred embodiment of the typing method of the present invention,the mixed cell culture is a mixture consisting of 80-99% of thepermissive cell line and 1-20% of the genetically engineered cell line.In yet other preferred embodiments, the cell types are in approximateequal proportions in the mixed cell cultures. As with the first methoddescribed, it is not intended that the present invention be limited toany particular herpesvirus. In one particular embodiment, the member ofthe herpesvirus family detected and typed using the method of thepresent invention is selected from the group comprising HSV-1, HSV-2,CMV, VZV, EBV, and human herpes viruses such as HHV-6, HHV-7, and HHV-8.It is intended that one or more herpesviruses may be detected and typedin one specimen. In this manner, co-infection with multipleherpesviruses may be diagnosed. For example, it is contemplated thatmixed infections with HSV-1 and HSV-2 may be detectable and theinfections distinguished using the methods of the present invention.

In one embodiment of the typing method, the reporter gene comprises E.coli lacZ gene. However, it is not intended that the reporter gene belimited to lacZ. Indeed, it is contemplated that any reporter gene maybe used in this method. In one particularly preferred embodiment, thedetection of the reporter gene is accomplished through the use ofhistochemical staining. It is contemplated that one member of theherpesvirus family will produce an histochemically pattern of expressionthat is distinguishable from the histochemical patterns produced byother members of the herpesvirus family. In this manner, it is possibleto use the methods of the present invention to distinguish infectionwith one herpesvirus from infection with another herpesvirus.

It is contemplated that the method of the present invention will be usedin conjunction with controls of known positivity and negativity for thevirus(es) of interest. Thus, it is contemplated that the pattern ofexpression present in a test sample (e.g., from a clinical specimen)will be compared to the patterns of expression in control samples knownto be positive and/or negative for the virus(es) of interest. It is alsocontemplated that effects unrelated to the expression of the reportergene will be detectable, including but not limited to CPE. Theseeffects, alone and in combination with reporter gene expression may beused to detect the presence of viral infection, as well as provideinformation to distinguish between viruses.

In yet another embodiment, the present invention provides a compositioncomprising a mixed cell culture, wherein the mixed cell culturecomprises the combination of a genetically engineered cell linetransformed with a promoter sequence from a virus, wherein the promotersequence is operably linked to a reporter gene, and wherein expressionof the reporter gene is dependent upon and quantitatively proportionalto the presence of virus, and a non-engineered cell line which ispermissive for virus infection.

In one embodiment of the composition, the mixed cell culture is amixture consisting of 1-20% of the genetically engineered cell line and80-99% of the permissive cell line. In yet other preferred embodiments,the cell types are in approximate equal proportions in the mixed cellcultures. In one preferred embodiment of the composition, thegenetically engineered cell line component may comprise a promoter for agene that encodes ribonucleotide reductase. In an alternative preferredembodiment, the promoter may comprise genes that encode one or moresubunits of ribonucleotide reductase. In one particularly preferredembodiment, the genetically engineered cell line is BHKICP10LacZ, whilein another particularly preferred embodiment, the genetically engineeredcell line is BHKICP6LacZ. In an alternative embodiment of thecomposition, the genetically engineered cell line comprises an E. colilacZ gene positioned 3′ to a virus inducible promoter. It iscontemplated that this lacZ gene be positioned immediately 3′ to thisvirus-inducible promoter. However, it is not intended that thesesequences will be contiguous. Indeed, it is contemplated only that thereporter and promoter genes are operably linked. Furthermore, it iscontemplated that the composition will comprise promoter sequences fromany virus, including but not limited to members of the herpesvirusfamily. It is also contemplated that the non-engineered cell line bepermissive for infection by any number of viruses, including but notlimited to members of the herpesvirus family.

In one preferred embodiment, the composition includes a geneticallyengineered cell line which includes a promoter for a gene that encodes aribonucleotide reductase large subunit and the virus is a member of theherpesvirus family selected from the group consisting of HSV-1, HSV-2,CMV, VZV, EBV, HHV-6, HHV-7, and HHV-8. However, it is not intended thatthe present invention be limited to any particular herpesvirus. In onepreferred embodiment, the genetically engineered cell line componentcontains an ICP10 promoter and the herpesvirus family member is HSV-2,while in another preferred embodiment, the genetically engineered cellline comprises an ICP6 promoter and the herpesvirus family member isHSV-1.

It is contemplated that the detection of reporter gene expression beaccomplished through the use of various methods, including, but notlimited to calorimetric, fluorimetric or luminometric assays or assaysystems. In one preferred embodiment, the reporter gene encodesβ-galactosidase.

In one embodiment, the composition includes a genetically engineeredcell line that is a mammalian cell line susceptible to infection byvirus. In one preferred embodiment, the genetically engineered cell linecomprises baby hamster kidney cells (e.g., cell lines derived from BHKcells). In one embodiment, the composition includes a permissive cellline that is permissive to infection by herpesviruses, including but notlimited to HSV-1 and HSV-2. In a particularly preferred embodiment, thepermissive cell line is MRC-5. It is not intended that the compositionof the present invention be limited to detection of viral infectionbased on the expression of the reporter gene, as effects such as CPE mayalso be detectable.

The present invention also provides a kit for assaying for the presenceof infectious herpesvirus in a specimen. The kit includes: a) a supplyof a mixed cell line comprised of a cell line of genetically engineeredmammalian cells susceptible to infection by herpesvirus, wherein thecell line contains an oligonucleotide having a sequence comprising avirus promoter sequence operably linked to a reporter gene, and wherethe expression of the reporter gene is dependent upon and quantitativelyproportional to the presence of virus in the specimen; and a cell linepermissive for virus; and b) a supply of reagents to detect theexpression of the reporter gene. It is not intended that the promotersequences present within the genetically engineered cell line be limitedto any particular virus or virus family. It is contemplated that anyvirus promoter will be useful in the kit of the present invention.However, in one preferred embodiment, herpesvirus promoter sequences arepresent in the genetically engineered cell line.

It is contemplated that various promoter sequences will be useful in thekit of the present invention. However, in a preferred embodiment, thepromoter encodes either a complete ribonucleotide reductase enzyme, orin the alternative, subunits of ribonucleotide reductase. In oneparticularly preferred embodiment, the promoter sequence contains apromoter for a gene that encodes a ribonucleotide reductase largesubunit and the herpesvirus is a herpesvirus family member selected fromthe group consisting of HSV-1, HSV-2, CMV, VZV, EBV, HHV-6, HHV-7, andHHV-8. However, it is not intended that the kit will be limited to thislist of herpesviruses. Indeed, it is contemplated that any herpesvirusmay be detected using the present kit. In one particularly preferredembodiment of the kit, the promoter sequence contains an ICP10 promoterand the herpesvirus family member is HSV-2, while in an alternativepreferred embodiment, the promoter sequence contains an ICP6 promoterand the herpesvirus family member is HSV-1.

In one preferred embodiment of the kit, the promoter sequence present inthe genetically engineered cell line comprises an E. coli lacZ gene thatis operably linked to a herpesvirus inducible promoter. In oneparticularly preferred embodiment, the genetically engineered mammaliancells are BHKICP10LacZ cells, while in an alternative embodiment, thecells are BHKICP6LacZ cells.

In one preferred embodiment, the reporter gene encodes β-galactosidase.However, it is not intended that the present invention be limited to anyparticular reporter gene. It is also contemplated that the reporter genewill encode any number of enzymes that are amenable to detection byvarious methods, including but not limited to such methods ascolorimetric, fluorimetric or luminometric assay systems. In onepreferred embodiment of the kit, the reagents provided for the detectionof reporter gene expression may include, but are not limited to,solutions of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside,o-nitrophenyl-galactopyranoside solution, and fluoresceindi-β-D-galactopyranoside. However, it is not intended to limit the kitto these assay systems, as other systems (e.g., radiometric assaysystems) may be useful.

It is contemplated that the kit of the present invention may alsoinclude additional components, such as materials suitable for positiveand negative controls and instructions for use. It is not intended thatthe kit of the present invention be limited to the mixed cell line andreagents for the detection of reporter gene expression. It is alsointended that the kit will be useful for detection of viral effects oncells other than and unrelated to reporter gene expression. For example,it is contemplated that the kit may be useful for detection of CPE.

DESCRIPTION OF THE INVENTION

The present invention generally relates to the field of diagnosticmicrobiology, and more particularly, to compositions and methods fordetecting and differentiating one or more viruses or other intracellularparasites present in a specimen. The present invention also providescompositions and methods to evaluate the susceptibility of a organismsto antimicrobial agents.

The present invention provides methods and compositions for thedetection of several different viruses, as well as other intracellularorganisms present in clinical and other specimens in a single cellculture unit comprised of a mixture of cells grown in a manner toco-exist as a monolayer of relatively equivalent ratio and demonstratingcomplementary susceptibilities to a wider range of viruses and/or otherorganisms than could be detected by each individual cell line. Forexample, the viral assays involve inoculating a cell mixture with aspecimen suspected of containing a virus, allowing a sufficient periodof time for the virus infectious cycle to proceed, followed by thedetection and/or quantification of the number of virus-infected cells todetermine the number of infectious virions in the specimen. Thisdetection step may be accomplished using any number of availableconfirmation methods, including specific viral antigen detection usingantigen-specific antibodies, nucleic acid probes, and reporter genedetection. The assay also provides reliable methods and compositions forthe quantification of the number of infectious virions present in asample. In addition, the methods and compositions of the presentinvention are sufficiently sensitive that the presence of a singlevirion in a specimen may be detected.

The present invention also provides compositions comprising novelmixtures of various cell types traditionally used in single cell assays.In preferred embodiments, the cells are mixed to produce mixed monolayercell cultures. One such mixed cell culture includes mink lung (e.g.,Mv1Lu) cells co-cultivated with human mucoepidermoid cells (e.g.,NCI-H292; also referred to as “H292” cells). This cell mixture issusceptible to viruses such as influenza A, influenza B, RSV,parainfluenza types 1, 2, and 3, adenovirus, and CMV (i.e., the group ofviruses most commonly associated with respiratory virus disease). Inother mixed cultures, buffalo green monkey kidney cells (BGMK) areco-cultivated with NCI-H292 cells for the detection and identificationof enteroviruses, such as poliovirus, echoviruses and Coxsackie B virus(e.g., Coxsackie A and B viruses), and numbered EV strains. In additionto enteroviruses, it is contemplated that the present inventionencompass cell types that are susceptible to picornaviruses such asHepatitis A.

The present invention also provides compositions comprising novelmixtures of different cell types traditionally used in single cellassays that are co-cultivated with genetically engineered cells. Inparticularly preferred embodiments, the genetically engineered cell lineis a DNA-transfected cell line that is susceptible to infection by avirus, the cell line having been stably transformed with a chimeric genecomprising a virus-inducible promoter and a gene coding for an enzyme,the expression of the enzyme being dependent upon the presence of thevirus. Such genetically engineered cells are, for example, described inU.S. Pat. No. 5,418,132, herein incorporated by reference. In onepreferred embodiment, a cell mixture includes human lung fibroblasts(e.g., MRC-5 cells) co-cultivated with a stable baby hamster kidney(BHK) cell line, the genome of which has been engineered to contain theE. coli lacZ gene behind (i.e., 3′ to) an inducible HSV promoter, HSV-1ICP6 promoter (BHK/ICP6LacZ-5 cells are available from the ATCC asCRL-12072). This cell mixture is susceptible to infection by CMV and HSVtypes 1 and 2.

In yet another embodiment, the present invention provides compositionscomprising novel mixtures of different types of genetically engineeredcells. In particularly preferred embodiments, the genetically engineeredcell line is a DNA-transfected cell line that is susceptible toinfection by a virus, the cell line having been stably transformed witha chimeric gene comprising a virus-inducible promoter and a gene codingfor an enzyme, the expression of the enzyme being dependent upon thepresence of the virus. The second genetically engineered cell line is aDNA-transfected cell line susceptible to viral infection, which isstably transformed with a chimeric gene comprising a virus-induciblepromoter, and a gene encoding a second enzyme (i.e., an enzyme that isdifferent from that associated with the first cell line), in which theexpression of the second enzyme is dependent upon the presence of asecond virus. In one preferred embodiment, a cell mixture is prepared inwhich engineered BHK cells (e.g., BHK/ICP6/LacZ-5 cells) areco-cultivated with a stable mink lung cell line (Mv1Lu), the genome ofwhich has been engineered to contain the an inducible CMV promoter (theCMV UL45 promoter); these cells are referred to as “MLID5” cells, andare disclosed in U.S. patent application Ser. No. 08/846,026, hereinincorporated by reference. This cell mixture is susceptible to infectionby CMV and HSV virus types 1 and 2 (HSV-1 and HSV-2), with CMV infectingthe genetically engineered BHK cells, and HSV-1 and HSV-2 preferentiallyinfecting the mink lung cells. In another embodiment, the presentinvention contemplates the use of genetically engineered cells (e.g.,mink lung cells) in which the cell genome is engineered to contain thefirefly luciferase gene behind (i.e., 3′ to) an inducible CMV promoter;these cells are also described in U.S. patent application Ser. No.08/846,026. However, it is not intended that the present invention belimited to any particular cell types or cell lines, nor is it intendedthat the present invention be limited to any particular combinations ofcells. It is also not intended that the present invention be limited interms of the genetically engineered cells.

The following Table provides a matrix which indicates the ability ofvarious cells to form single, confluent monolayers, as well asco-cultivated, confluent, mixed cell monolayers.

TABLE 1 Cell Cultures NCI- MRC-5 CV-1 BGMK McCoy BHK* A549 HEp-2 Mv1LuH292 1 2 3 4 5 6 7 8 9 MRC-5 A + + + No + + + + + + CV-1 B + +No + + + + + + BGMK C + + + + + + + + McCoy D + + + + Yes + + BHK* E+ + + + + + A549 F + + + + + HEp-2 G + + + + Mv1Lu H + + + NCI- I + +H292 + + Denotes single cell types producing confluent monolayers +Denotes some degree of dimorphic, mixed monolayer Yes Denotes cellmixtures that appear very uniform, with an even distribution No Denotescell mixtures that did not appear to work * Denotes geneticallyengineered ELVIS BHK cells.

In yet another embodiment, the present invention provides kits forassaying samples for the presence of infectious viruses. In these kits,mixed cell cultures are provided which facilitate the detection andidentification of particular virus groups (e.g., viruses associated withrespiratory infections/diseases). In the kits, co-cultivated cells aresupplied either frozen or dispensed (i.e., ready for use) in shellvials, tubes, or multiwell plates. These cells are susceptible toinfection by the virus group of interest as indicated by the sampletype. In preferred embodiments, the kits also include reagents necessaryto detect expression of viral antigens or virus-induced reporter geneexpression.

One of the several advantages of the present invention is that itprovides rapid and sensitive assay systems for the detection andidentification of a single virus type from a multiplicity ofpossibilities, in a single mixed cell unit that is suitable fordiagnostic assay. Thus, the present invention eliminates the need formultiple cell lines cultured in individual containers, provides reliableresults in 1-3 days following inoculation of the cell cultures, ratherthan 1-28 days, eliminates the necessity of working with primary cellcultures, provides an efficient screening method for grouping andpreliminary identification of viruses, and provides assay systems thatare highly specific for viruses capable of inducing reporter geneexpression. Thus, the present invention clearly fulfills a need that hasbeen heretofore unmet in the field of diagnostic virology.

DEFINITIONS

The terms “sample” and “specimen” in the present specification andclaims are used in their broadest sense. On the one hand, they are meantto include a specimen or culture. On the other hand, they are meant toinclude both biological and environmental samples. These termsencompasses all types of samples obtained from humans and other animals,including but not limited to, body fluids such as urine, blood, fecalmatter, cerebrospinal fluid (CSF), semen, sputum, and saliva, as well assolid tissue. These terms also refers to swabs and other samplingdevices which are commonly used to obtain samples for culture ofmicroorganisms.

Biological samples may be animal, including human, fluid or tissue, foodproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Environmental samples include environmentalmaterial such as surface matter, soil, water, and industrial samples, aswell as samples obtained from food and dairy processing instruments,apparatus, equipment, disposable, and non-disposable items. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

Whether biological or environmental, a sample suspected of containingmicroorganisms may (or may not) first be subjected to an enrichmentmeans to create a “pure culture” of microorganisms. By “enrichmentmeans” or “enrichment treatment,” the present invention contemplates (i)conventional techniques for isolating a particular microorganism ofinterest away from other microorganisms by means of any culture mediumand/or technique, and (ii) novel techniques for isolating particularmicroorganisms away from other microorganisms. It is not intended thatthe present invention be limited only to one enrichment step or type ofenrichment means. For example, it is within the scope of the presentinvention, following subjecting a sample to a conventional enrichmentmeans, to subject the resultant preparation to further purification suchthat a pure culture of a strain of a species of interest is produced.This pure culture may then be analyzed by the medium and method of thepresent invention.

As used herein, the term “organism” and “microorganism,” are used torefer to any species or type of microorganism, including but not limitedto viruses and bacteria, including rickettsia and chlamydia. Thus, theterm encompasses, but is not limited to DNA and RNA viruses, as well asorganisms within the orders Rickettsiales and Chlamydiales.

As used herein, the term “culture,” refers to any sample or specimenwhich is suspected of containing one or more microorganisms. “Purecultures” are cultures in which the organisms present are only of onestrain of a particular genus and species. This is in contrast to “mixedcultures,” which are cultures in which more than one genus and/orspecies of microorganism are present.

As used herein, the term “cell type,” refers to any cell, regardless ofits source or characteristics.

As used herein, the term “cell line,” refers to cells that are culturedin vitro, including primary cell lines, finite cell lines, continuouscell lines, and transformed cell lines.

As used herein, the terms “primary cell culture,” and “primary culture,”refer to cell cultures that have been directly obtained from animal orinsect tissue. These cultures may be derived from adults as well asfetal tissue.

As used herein, the term “finite cell lines,” refer to cell culturesthat are capable of a limited number of population doublings prior tosenescence.

As used herein, the term “continuous cell lines,” refer to cell culturesthat have undergone a “crisis” phase during which a population of cellsin a primary or finite cell line apparently ceases to grow, but yet apopulation of cells emerges with the general characteristics of areduced cell size, higher growth rate, higher cloning efficiency,increased tumorigenicity, and a variable chromosomal complement. Thesecells often result from spontaneous transformation in vitro. These cellshave an indefinite lifespan.

As used herein, the term “transformed cell lines,” refers to cellcultures that have been transformed into continuous cell lines with thecharacteristics as described above. Transformed cell lines can bederived directly from tumor tissue and also by in vitro transformationof cells with whole virus (e.g., SV40 or EBV), or DNA fragments derivedfrom a transforming virus using vector systems.

As used herein, the term “hybridomas,” refers to cells produced byfusing two cell types together. Commonly used hybridomas include thosecreated by the fusion of antibody-secreting B cells from an immunizedanimal, with a malignant myeloma cell line capable of indefinite growthin vitro. These cells are cloned and used to prepare monoclonalantibodies.

As used herein, the term “mixed cell culture,” refers to a mixture oftwo types of cells. In some preferred embodiments, the cells are celllines that are not genetically engineered, while in other preferredembodiments the cells are genetically engineered cell lines. In someembodiments, the one or more of the cell types is re “permissive” (i.e.,virus is capable of replication and spread from cell to cell within theculture). The present invention encompasses any combination of celltypes suitable for the detection, identification, and/or quantitation ofviruses in samples, including mixed cell cultures in which all of thecell types used are not genetically engineered, mixtures in which one ormore of the cell types are genetically engineered and the remaining celltypes are not genetically engineered, and mixtures in which all of thecell types are genetically engineered.

As used herein, the term “suitable for the detection of intracellularparasites,” refers to cell cultures that can be successfully used todetect the presence of an intracellular parasite in a sample. Inpreferred embodiments, the cell cultures are capable of maintainingtheir susceptibility to infection and/or support replication of theintracellular parasite. It is not intended that the present invention belimited to a particular cell type or intracellular parasite.

As used herein, the term “susceptible to infection” refers to theability of a cell to become infected with virus or another intracellularorganism. Although it encompasses “permissive” infections, it is notintended that the term be so limited, as it is intended that the termencompass circumstances in which a cell is infected, but the organismdoes not necessarily replicate and/or spread from the infected cell toother cells. The phrase “viral proliferation,” as used herein describesthe spread or passage of infectious virus from a permissive cell type toadditional cells of either a permissive or susceptible character.

As used herein, the terms “monolayer,” “monolayer culture,” and“monolayer cell culture,” refer to cells that have adhered to asubstrate and grow in as a layer that is one cell in thickness.Monolayers may be grown in any format, including but not limited toflasks, tubes, coverslips (e.g., shell vials), roller bottles, etc.Cells may also be grown attached to microcarriers, including but notlimited to beads.

As used herein, the term “suspension,” and “suspension culture,” refersto cells that survive and proliferate without being attached to asubstrate. Suspension cultures are typically produced usinghematopoietic cells, transformed cell lines, and cells from malignanttumors.

As used herein, the terms “culture media,” and “cell culture media,”refers to media that are suitable to support the growth of cells invitro (i.e., cell cultures). It is not intended that the term be limitedto any particular culture medium. For example, it is intended that thedefinition encompass outgrowth as well as maintenance media. Indeed, itis intended that the term encompass any culture medium suitable for thegrowth of the cell cultures of interest.

As used herein, the term “obligate intracellular parasite,” (or“obligate intracellular organism) refers to any organism which requiresan intracellular environment for its survival and/or replication.Obligate intracellular parasites include viruses, as well as many otherorganisms, including certain bacteria (e.g., most members of the ordersRickettsiales [e.g., Coxiella, Rickettsia and Ehrlichia] andChlamydiales [e.g., C. trachomatis, C. psittaci], etc). The term“intracellular parasite,” refers to any organism that may be foundwithin the cells of a host animal, including but not limited to obligateintracellular parasites briefly described above. For example,intracellular parasites include organisms such as Brucella, Listeria,Mycobacterium (e.g., M. tuberculosis and M. leprae), and Plasmodium, aswell as Rochalimea.

As used herein, the term “antimicrobial,” is used in reference to anycompound which inhibits the growth of, or kills microorganisms. It isintended that the term be used in its broadest sense, and includes, butis not limited to compounds such as antibiotics which are producednaturally or synthetically. It is also intended that the term includescompounds and elements that are useful for inhibiting the growth of, orkilling microorganisms.

As used herein, the terms “chromogenic compound,” and “chromogenicsubstrate,” refer to any compound useful in detection systems by theirlight absorption or emission characteristics. The term is intended toencompass any enzymatic cleavage products, soluble, as well asinsoluble, which are detectable either visually or with opticalmachinery. Included within the designation “chromogenic” are allenzymatic substrates which produce an end product which is detectable asa color change. This includes, but is not limited to any color, as usedin the traditional sense of “colors,” such as indigo, blue, red, yellow,green, orange, brown, etc., as well as fluorochromic or fluorogeniccompounds, which produce colors detectable with fluorescence (e.g., theyellow-green of fluorescein, the red of rhodamine, etc.). It is intendedthat such other indicators as dyes (e.g., pH) and luminogenic compoundsbe encompassed within this definition.

As used herein, the commonly used meaning of the terms “pH indicator,”“redox indicator,” and “oxidation-reduction indicator,” are intended.Thus, “pH indicator,” encompasses all compounds commonly used fordetection of pH changes, including, but not limited to phenol red,neutral red, bromthymol blue, bromcresol purple, bromcresol green,bromchlorophenol blue, m-cresol purple, thymol blue, bromcresol purple,xylenol blue, methyl red, methyl orange, and cresol red. The terms“redox indicator,” and “oxidation-reduction indicator,” encompasses allcompounds commonly used for detection of oxidation/reduction potentials(i.e., “eH”) including, but not limited to various types or forms oftetrazolium, resazurin, and methylene blue.

As used herein, the term “inoculating suspension,” or “inoculant,” isused in reference to a suspension which may be inoculated with organismsto be tested. It is not intended that the term “inoculating suspension,”be limited to a particular fluid or liquid substance. For example,inoculating suspensions may be comprised of water, saline, or an aqueoussolution. It is also contemplated that an inoculating suspension mayinclude a component to which water, saline or any aqueous material isadded. It is contemplated in one embodiment, that the componentcomprises at least one component useful for the intended microorganism.It is not intended that the present invention be limited to a particularcomponent.

As used herein, the term “kit,” is used in reference to a combination ofreagents and other materials.

As used herein, the term “primary isolation,” refers to the process ofculturing organisms directly from a sample. As used herein, the term“isolation,” refers to any cultivation of organisms, whether it beprimary isolation or any subsequent cultivation, including “passage,” or“transfer,” of stock cultures of organisms for maintenance and/or use.

As used herein, the term “presumptive diagnosis,” refers to apreliminary diagnosis which gives some guidance to the treatingphysician as to the etiologic organism involved in the patient'sdisease. Presumptive diagnoses are often based on “presumptiveidentifications,” which as used herein refer to the preliminaryidentification of a microorganism.

As used herein, the term “definitive diagnosis,” is used to refer to afinal diagnosis in which the etiologic agent of the patient's diseasehas been identified. The term “definitive identification” is used inreference to the final identification of an organism to the genus and/orspecies level.

The term “recombinant DNA molecule,” as used herein refers to a DNAmolecule which is comprised of segments of DNA joined together by meansof molecular biological techniques.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides is referred to as the“5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or 5′ of the “downstream” or 3′ elements. Thisterminology reflects the fact that transcription proceeds in a 5′ to 3′fashion along the DNA strand. The promoter and enhancer elements whichdirect transcription of a linked gene are generally located 5′ orupstream of the coding region (enhancer elements can exert their effecteven when located 3′ of the promoter element and the coding region).Transcription termination and polyadenylation signals are located 3′ ordownstream of the coding region.

The term “an oligonucleotide having a nucleotide sequence encoding agene,” refers to a DNA sequence comprising the coding region of a geneor, in other words, the DNA sequence which encodes a gene product. Thecoding region may be present in either a cDNA or genomic DNA form.Suitable control elements such as enhancers, promoters, splicejunctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the vectors ofthe present invention may contain endogenous enhancers and/or promoters,splice junctions, intervening sequences, polyadenylation signals, etc.or a combination of both endogenous and exogenous control elements.

The term “transcription unit,” as used herein refers to the segment ofDNA between the sites of initiation and termination of transcription andthe regulatory elements necessary for the efficient initiation andtermination. For example, a segment of DNA comprising anenhancer/promoter, a coding region, and a termination andpolyadenylation sequence comprises a transcription unit.

The term “regulatory element,” as used herein refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. (defined infra).

The terms “reporter gene construct,” or “reporter gene vector,” as usedherein refers to a recombinant DNA molecule containing a sequenceencoding the product of a reporter gene and appropriate nucleic acidsequences necessary for the expression of the operably linked codingsequence in a particular host organism. Eukaryotic cells are known toutilize promoters, enhancers, and termination and polyadenylationsignals.

The term “reporter gene,” refers to an oligonucleotide having a sequenceencoding a gene product (typically an enzyme) which is easily andquantifiably assayed when a construct comprising the reporter genesequence operably linked to a heterologous promoter and/or enhancerelement is introduced into cells containing (or which can be made tocontain) the factors necessary for the activation of the promoter and/orenhancer elements. Examples of reporter genes include but are notlimited to bacterial genes encoding β-galactosidase (lacZ), thebacterial chloramphenicol acetyltransferase (cat) genes, fireflyluciferase genes and genes encoding β-glucuronidase (GUS).

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (Maniatis, et al., Science 236:1237 [1987]). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells and viruses(analogous control elements [i.e., promoters, are also found inprokaryotes]). The selection of a particular promoter and enhancerdepends on what cell type is to be used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types (forreview see Voss, et al., Trends Biochem. Sci., 11:287 [1986], andManiatis, et al., supra [1987]). For example, the SV40 early geneenhancer is very active in a wide variety of cell types from manymammalian species and has been widely used for the expression ofproteins in mammalian cells (Dijkema, et al., EMBO J. 4:761 [1985]). Twoother examples of promoter/enhancer elements active in a broad range ofmammalian cell types are those from the human elongation factor 1α gene(Uetsuki et al., J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene91:217 [1990]; and Mizushima and Nagata, Nuc. Acids. Res., 18:5322[1990]) and the long terminal repeats of the Rous sarcoma virus (Gormanet al., Proc. Natl. Acad. Sci. USA 79:6777 [1982]), and the humancytomegalovirus (Boshart et al., Cell 41:521 [1985]).

The term “promoter/enhancer,” denotes a segment of DNA which containssequences capable of providing both promoter and enhancer functions (forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions). The enhancer/promoter may be “endogenous,” or“exogenous,” or “heterologous.” An endogenous enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An exogenous(heterologous) enhancer/promoter is one which is placed in juxtapositionto a gene by means of genetic manipulation (i.e., molecular biologicaltechniques).

The presence of “splicing signals,” on an expression vector oftenresults in higher levels of expression of the recombinant transcript.Splicing signals mediate the removal of introns from the primary RNAtranscript and consist of a splice donor and acceptor site (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York [1989], pp. 16.7-16.8). A commonly usedsplice donor and acceptor site is the splice junction from the 16S RNAof SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires signals directing the efficient termination and polyadenylationof the resulting transcript. Transcription termination signals aregenerally found downstream of the polyadenylation signal and are a fewhundred nucleotides in length. The term “poly A site,” or “poly Asequence,” as used herein denotes a DNA sequence which directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous poly A signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous poly A signal is one which is isolated from onegene and placed 3′ of another gene. A commonly used heterologous poly Asignal is the SV40 poly A signal. The SV40 poly A signal is contained ona 237 bp BamHI/BclI restriction fragment and directs both terminationand polyadenylation (Sambrook, supra, at 16.6-16.7). This 237 bpfragment is contained within a 671 bp BamHI/PstI restriction fragment.

The term “genetically engineered cell line,” refers to a cell line thatcontains heterologous DNA introduced into the cell line by means ofmolecular biological techniques (i.e., recombinant DNA technology).

The term “stable transfection,” or “stably transfected,” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant,” refers to a cell whichhas stably integrated foreign DNA into the genomic DNA.

The term “stable transfection” (or “stably transfected”), refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant,” refers to a cell whichhas stably integrated foreign DNA into the genomic DNA.

The term “selectable marker,” as used herein refers to the use of a genewhich encodes an enzymatic activity that confers resistance to anantibiotic or drug upon the cell in which the selectable marker isexpressed. Selectable markers may be “dominant”; a dominant selectablemarker encodes an enzymatic activity which can be detected in anymammalian cell line. Examples of dominant selectable markers include thebacterial aminoglycoside 3′ phosphotransferase gene (also referred to asthe neo gene) which confers resistance to the drug G418 in mammaliancells, the bacterial hygromycin G phosphotransferase (hyg) gene whichconfers resistance to the antibiotic hygromycin and the bacterialxanthine-guanine phosphoribosyl transferase gene (also referred to asthe gpt gene) which confers the ability to grow in the presence ofmycophenolic acid. Other selectable markers are not dominant in thattheir use must be in conjunction with a cell line that lacks therelevant enzyme activity. Examples of non-dominant selectable markersinclude the thymidine kinase (tk) gene which is used in conjunction withtk⁻ cell lines, the CAD gene which is used in conjunction withCAD-deficient cells and the mammalian hypoxanthine-guaninephosphoribosyl transferase (hprt) gene which is used in conjunction withhprt⁻ cell lines. A review of the use of selectable markers in mammaliancell lines is provided in Sambrook et al., supra at pp.16.9-16.15.

The terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and“DNA encoding,” refer to the order or sequence of deoxyribonucleotidesalong a strand of deoxyribonucleic acid. The order of thesedeoxyribonucleotides determines the order of amino acids along thepolypeptide (protein) chain. The DNA sequence thus codes for the aminoacid sequence.

The terms “confluent” or “confluency” as used herein in reference to anadherent cell line define a condition wherein cells throughout a cultureare in contact with each other creating what appears to be a continuoussheet or “monolayer” of cells.

The terms “cytopathic effect” or “CPE” as used herein describe changesin cellular structure (i.e., a pathologic effect) resulting fromexternal agents such viruses. Common cytopathic effects include celldestruction, syncytia (i.e., fused giant cells) formation, cell roundingvacuole formation, and formation of inclusion bodies. CPE results fromactions of a virus on permissive cells that negatively affect theability of the permissive cellular host to preform its requiredfunctions to remain viable. In in vitro cell culture systems, CPE isevident when cells, as part of a confluent monolayer, show regions ofnon-confluence after contact with a specimen that contains a virus. Theobserved microscopic effect is generally focal in nature and the foci isinitiated by a single virion. However, depending upon viral load in thesample, CPE may be observed throughout the monolayer after a sufficientperiod of incubation. Cells demonstrating viral induced CPE usuallychange morphology to a rounded shape, and over a prolonged period oftime can die and be released form their anchorage points in themonolayer. When many cells reach the point of focal destruction, thearea is called a viral plaque, which appears as a hole in the monolayer.Cytopathic effects are readily discernable and distinguishable by thoseskilled in the art.

The abbreviation “ONPG,” represents o-Nitrophenyl-β-D-Galactopyranoside.ONPG is a substrate for the enzyme β-galactosidase (β-gal). The reactionbetween ONPG and β-gal produces a yellow product which can be quantifiedspectrophotometrically at 405 nm.

The abbreviation “X-gal,” represents the chemical compound5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, a substrate for theenzyme β-galactosidase. The reaction between X-gal and β-galactosidaseresults in the formation of a blue precipitate which is visuallydiscernable.

The term “hybriwix,” represents a product of Diagnostic Hybrids, Inc.,Athens, Ohio which allows for quantification of certain viral DNA in aninfected monolayer of cells by DNA hybridization. “DNA hybridization” isthe annealing of two complementary DNA molecules whose base sequencesmatch according to the rules of base pairing. DNA hybridization is usedto identify or quantify an unknown or “target” DNA by hybridization to aknown DNA or “probe.” The probe is typically labeled with a reportermolecule such as ¹²⁵I, a radioisotope which can be detected andquantified with a gamma counter.

The phrase “plaque reduction assay,” or “PRA,” as used herein describesa standard method used to determine efficacy of anti-viral drugs byenumerating a decrease in plaque formation in a cell monolayer exposedto a drug. A “plaque” is a defined area of “CPE.” It is usually theresult of infection of the cell monolayer with a single infectious viruswhich then replicates and spreads to adjacent cells of the monolayer. Aplaque may also be referred to as a “focus of viral infection.”

The term “permissive” as used herein describes the sequence ofinteractive events between a virus and its putative host cell. Theprocess begins with viral adsorption to the host cell surface and endswith release of infectious virions. A cell is “permissive” if it readilypermits the spread of virus to other cells. Many methods are availablefor the determination of the permissiveness of a given cell line,including, but not limited to plaque reduction assays, comparisons ofthe production and/or quantitation of viral proteins based on resultsobtained from gel electrophoresis, relative comparisons usinghybridization analysis to analyze DNA or RNA content, etc.

The term “susceptible,” as used herein describes the extent that apermissive or non-permissive host cell can adsorb and be penetrated by avirus. A cell line may be susceptible without being permissive in thatit can be penetrated but not release virions. A permissive cell linehowever must be susceptible.

The phrase “seed on,” as used herein describes the act of transferringan aqueous solution of suspended cells into a vessel containing cellsadhered to a surface, after which the vessel is stored for a sufficientperiod of time to allow the suspended cells or “seeds” to settle out bygravity and attach in a relatively uniform manner to the adhered cellsand become integrated into the final cell monolayer as a mixture. A“mixed cell monolayer,” results from the “seed on” process.

The phrase “seed in,” as used herein describes the mixing of two or moreaqueous solutions of suspended tissue culture cells, each cellsuspension having different cellular properties, and transfer of suchmixture of cells into a vessel which is stored for a sufficient periodof time to allow the suspended cells to settle out by gravity and attachin a relatively uniform manner such that the distribution of any singlecell type is indicative of the relative ratio of the cells in theoriginal mixture.

The term “starts,” as used herein refers to the reporter cells whichrepresent a primary infection of virus. The virus infects a reportercell (a genetically engineered cell) and induces the expression of thereporter gene. A reporter cell can be non-permissive (i.e.permissiveness of the reporter cells is not required) and still producestarts.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ×g(times gravity); ° C. (degrees Centigrade); FBS (fetal bovine serum);PBS (phosphate buffered saline; HEPES(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]); HBSS (Hank'sBalanced Salt Solution); MEM (Minimal Essential Medium); EMEM (Eagle'sMinimal Essential Medium); BBL (Becton Dickinson Microbiology Systems,Cockeysville, Md.); DIFCO (Difco Laboratories, Detroit, Mich.); U.S.Biochemical (U.S. Biochemical Corp., Cleveland, Ohio); Chemicon(Chemicon, Inc., Temecula, Calif.); Dako (Dako Corporation, Carpinteria,Calif.); Fisher (Fisher Scientific, Pittsburgh, Pa.); Sigma (SigmaChemical Co., St. Louis, Mo.); ATCC (American Type Culture Collection,Rockville, Md.); Bartel's (Bartels, Issaquah, Wash.); and BioWhittaker(BioWhittaker, Walkersville, Md.).

The cells used during the development of the present invention anddescribed in the following Examples, were obtained from the ATCC, withthe exception being that the BGMK and PRMK cells were obtained fromBioWhittaker, and the MRC-5 cells were obtained from both ATCC andBioWhittaker. The ATCC numbers of the cells are indicated in thefollowing Table.

TABLE 2 ATCC Cell Lines Cell Line ATCC Number BHK/ICP6LacZ-5 CCL-12072A549 CCL-185 CV-1 CCL-70 HEp-2 CCL-23 hs27 HFF; CRL-1634 Mv1Lu CCL-64McCoy CCL-1696 NCI-H292 CCL-1848 MRC-5 CCL-171 WI-38 CCL-75 Vero CCL-81MDCK (NBL-2) CCL-34 BHK21 CCL-10 HEL299 CCL-137 HeLa CCL-2

EXAMPLE 1 Co-Cultivation of Cells

In this Example, mixed cell cultures were established in which single,dimorphic cell sheets were produced at confluency.

In these experiments, all of the cell lines were cultured to confluencyin sterile polystyrene flasks in EMEM (Eagle's Minimal Essential Medium)with 25 mM HEPES, 7% fetal bovine serum (FBS), 2 mM L-glutamine, andpenicillin/streptomycin (100 Units/100 μg per ml of medium each).

Cells to be cultured were harvested by first rinsing source cellmonolayers with Hank's Balanced Salt Solution (HBSS) without magnesiumor calcium. Depending upon the cell line, the cells were dissociated byadding trypsin (0.125% in HBSS, without calcium or magnesium) ortrypsin-EDTA (0.25% in 1 mM EDTA in HBSS, without calcium or magnesium),or directly to the cell monolayer, and incubating for approximately 5minutes at ambient temperature. Ten volumes of cell culture medium wasadded to each trypsinized cell suspension and the cells were repeatedlypipetted in order to produce near-single cell suspensions (i.e., withoutcell aggregates). Each trypsinized cell suspension was diluted in anadequate volume of culture medium to produce an optical density of cellsuspension suitable to produce a confluent monolayer of cells within 2-3days of incubation in a 96-well microtiter plate. For single cellmonolayers (i.e., one cell type per well), 0.2 ml of suspension was usedto inoculate each well. For example, the final cell preparations rangedfrom a final optical density at 500 nm of 0.012 OD units/ml for CV-1cells to 0.03 OD units/ml for HEp-2 cells.

Cell mixture monolayers were produced by co-planting two distinct celltypes at an equal volume of each diluted cell suspension (i.e., 0.1 mlof each cell type was used to inoculate each well of a 96-wellmicrotiter plate). The cells were allowed to attach to the well surfaceby gravity for 30-60 minutes, and the inoculated microtiter plates wereincubated for up to three days at 36° C. in 5% CO₂ with 95% relativehumidity.

Periodically during incubation, single and mixed monolayers were checkedfor overall viability. The mixed cell culture monolayers were alsochecked for the ability of the cell lines to co-exist and develop as asingle cell sheet (i.e., a single monolayer), with two distinct cellmorphologies (i.e., dimorphic cell sheets), at an approximately equaldensity of each cell type. At confluency, the cells were treated with amethylene blue staining solution to fix the cells and stain them a lightblue in order to provide contrast for visualization using lightmicroscopy.

Some of the mixed monolayers successfully grew as a mixed cell monolayeradhered to the well surfaces, exhibiting a smooth, evenly distributedmonolayer. These mixed cultures were designated as “morphologic category1.” In these cultures, each cell type could be easily distinguished andappeared to survive well in a mixed monolayer, giving the appearance ofa single cell distribution. Mixed monolayers composed of HEp-2 and McCoycells displayed this morphology.

Some of the mixed monolayers successfully grew as a mixed monolayeradhered to the well surfaces, but exhibited two distinct morphologies atconfluency. These mixed cultures were designated as “morphologiccategory 2.” In these cultures, separate, distinct patches of each cellline co-existed within the monolayer, giving the appearance of oilmixing with water. Although an understanding of the mechanism is notnecessary in order to use the present invention, it is likely that thisappearance is most likely the result of contact inhibition between twospecific cell types. The relative sizes of the patches was foundprimarily to be a function of how evenly the cells were distributed atcell planting. The more even the cell distribution at planting, thepatches or islands were smaller as the monolayer reached confluency.Examples of monolayers that produced this appearance were mink lungcells co-cultivated with NCI-H292 cells, mink lung cells co-cultivatedby buffalo green monkey kidney (BGMK) cells, and human lung carcinomaA549 cells co-cultivated with NCI-H292 cells.

However, some cells types could not produce a mixed cell monolayer, whenmixed at relatively equal cell numbers at planting in the same culturemedium. In some of these cultures, only one of the cell types was foundto be viable (i.e., the culture was effectively a single cell type).Examples of mixed cell cultures that were found to be unsuitable for theproduction of mixed monolayers include human embryonic lung fibroblasts(MRC-5 cells) co-cultivated with BGMK cells. In this mixture, the MRC-5cells become toxic and form aggregates of dead cells soon afterplanting. Thus, at confluency, the monolayer only contains onefunctional, viable cell type, the BGMK cells. Thus, this cell mixturewas found to be unsuitable for producing mixed cell monolayers as thecells failed to form mixed cell monolayers of either a smooth ordimorphic morphologic type.

EXAMPLE 2 Detection of Respiratory Viruses in Mixed Cell Cultures

In this Example, mixed cell cultures were used to detect variousrespiratory viruses including Influenza A, RSV, adenoviruses,parainfluenza viruses, and Influenza B, present in clinical specimens.The mixed cells used in these experiments were Mv1Lu (mink lung cells)and NCI-H292 (human mucoepidermoid cells).

Cell Lines

Confluent T-225 flasks of Mv1Lu and H292 cells were prepared in EMEMwith HEPES, 10% FBS, 2 mM L-glutamine, and 50 μg/ml gentamicin. Thecells were harvested by first rinsing them in 30 ml HBSS withoutmagnesium and calcium. The cells were then dissociated from the flask bybrief exposure (i.e., until the cells lifted from the bottom of theflask) to 7 ml trypsin-EDTA solution as described in Example 1. Then, 30ml media was added to the cells to -prepare a cell suspensionconcentrate. The optical density of each cell suspension was determinedat 500 nm, using 3 ml of cells. Typically, the OD reading was 0.2/ml forboth the Mv1Lu and H292 cells. In addition to the Mv1Lu and H292 cells,rhesus monkey kidney cells (PRMK), A549 cells, and MDCK cells were usedin the present Example. These additional cell lines were prepared insingle cell cultures as known in the art.

Mixed Cell Cultures

When each cell suspension concentrate was determined to be 0.2 ODunits/ml, 5.2 ml of the Mv1Lu, and 8.7 ml H292 cell suspensions wereadded to 86.1 ml of culture medium, in order to provide an acceptableworking ratio of each cell type (i.e., it was a preparation of dilutedmixed cells). This ratio was devised in order to achieve a confluentmonolayer, in which each cell type covered a substantially equivalentsurface area within 1-3 days post-planting of the diluted mixed cells.Prior to dispensing, care was taken to prepare homogenous suspensions ofdiluted mixed cells. The mixed cells were dispensed at 0.75 ml per glassshell vial (i.e., glass vial containing a sterile glass coverslip).After planting, the vials were allowed to sit for 60 minutes at ambienttemperature so that the cells could settle by gravity and produce a moreoptimum cell distribution of each cell type. The mixed cells were thenincubated for 1-3 days at 36° C. in 5% CO₂, at 95% relative humidity.Subsequently, the shell vials were stored at ambient temperature tomaintain each cell type at substantively equivalent surface ratios forup to days from achieving confluency.

Samples and Processing

Nasopharyngeal specimens submitted to a diagnostic virology laboratorywere obtained from patients exhibiting influenza-like symptoms. Thespecimens were centrifuged to produce a cell pellet for direct antigentesting, and a specimen supernatant for inoculation of various cellcultures. The cell pellet was resuspended in phosphate buffer to preparea cell suspension and 25 μl portions of the cell suspension were spottedonto a glass slide and dried. Each spot of cells on the slide were thenfixed with fixative (e.g., acetone), and incubated for 30 minutes withindividual antibody solutions (Bartel's) capable of recognizing variousrespiratory viruses, including influenza A and RSV, as well as otherrespiratory viruses. A second antibody solution containing fluorescein(FITC) labelled goat anti-mouse antibodies and counterstain (Bartel's)was added to cover each cell spot on the slides, and incubated for anadditional 30 minutes at 35-37° C. The counterstain in the FITC-goatanti-mouse antibody solution contains Evans Blue, which stains the cellsand appears red under fluorescence. Slides prepared from thenasopharyngeal specimens were observed for positive (i.e.,virus-infected), apple green staining fluorescent cells, usingepifluorescence at 100-400×magnification.

In addition, 0.2 ml aliquots of the specimen supernatant were inoculatedonto various cell cultures prepared in shell vials containing glasscoverslips. The cell cultures included primary rhesus monkey kidneycells (PRMK; ViroMed or BioWhittaker), Mv1Lu cells (Diagnostic Hybrids)HEp-2 cells (Diagnostic Hybrids), MDCK, A549, and H292 cells, as singlecell monolayers, as well as mixed cell monolayers of Mv1Lu and H292cells, produced as described above.

Each inoculated shell vial was centrifuged for 60 minutes at 700×g, andthen incubated for 1-3 days at 36° C., in appropriate culture medium(e.g., EMEM containing 0.5 to 2% FBS, 2 mM L-glutamine, andpenicillin/streptomycin [100 Units/100 μg per ml of medium each]). Afterincubation, the culture medium was decanted, and the cells were fixed tothe glass coverslip with a solution of acetone and methanol (50:50,v/v). An antibody solution (Chemicon or Bartel's) containing a pool ofmonoclonal antibodies to multiple respiratory viruses, includingInfluenza A and RSV, as well as other respiratory viruses was added tocover each coverslip. The coverslips were then incubated for 30 minutesat 35-37° C. The antibody solution was then removed and the coverslipswere rinsed with PBS. A second antibody solution containing fluorescein(FITC) labelled goat anti-mouse antibodies and counterstain (Bartel's)was added to cover each coverslip, and incubated for an additional 30minutes at 35-37° C. The counterstain in the FITC-goat anti-mouseantibody solution contains Evans Blue, which stains the cells andappears red under fluorescence. Shell vial coverslips prepared from thenasopharyngeal specimens (i.e., inoculated cultures) were observed forpositive (i.e., virus-infected), apple green staining, fluorescentcells, using epifluorescence at 100-400×magnification.

Results

Some specimens demonstrated a positive direct antigen reaction on thecell spot incubated with Influenza A monoclonal antibody. Thesespecimens also demonstrated fluorescent staining on the single cellMv1Lu coverslip and the Mv1Lu/H292 mixed cell coverslip, but no or verylittle fluorescence on the single cell H292 coverslip. The H292 cellsare either not susceptible to this strain of Influenza A, or aresignificantly less susceptible, such that infection is not detectable.Additionally, in some cases (i.e., in specimens with low virus titers),the culture systems were more sensitive than the direct antigendetection method. Also, while the single PRMK cell cultures (i.e., the“gold standard” cells used to detect Influenza A) were positive for thepresence of Influenza A, with many specimens, the numbers of infectedcells and the total of number of positive specimens were lower thanthose identified as positive by the mixed cell monolayers.

In addition, both the MDCK and PRMK cells missed one low titer specimenpositive for Influenza A by direct antigen testing (IFA), and one otherspecimen that was also positive for Influenza A by IFA, while the Mv1Lucells detected the virus in all of the samples determined to be positivebased on direct antigen detection (IFA).

Some specimens demonstrated a positive direct antigen reaction on thecell spot incubated with RSV monoclonal antibody. These specimens alsodemonstrated fluorescent staining on the single cell H292 coverslip andthe MV1Lu/H292 mixed cell coverslip, but no or very little fluorescenceon the single cell MVILu coverslip. H292 cells are susceptible to RSVinfection, while Mv1Lu cells are not susceptible (or are significantlyless susceptible, such that infection is not detectable). In addition tothe mixed cell cultures, HEp-2 cells (i.e., the “gold standard” cellsused to detect RSV) were also observed for the presence of RSV; theperformance of HEp-2 cells was generally less sensitive than that of theMv1Lu and H292 mixed cell monolayers, or the H292 single cellmonolayers. These results with Influenza A tested in mink lung cells wasvery surprising, as the detection of Influenza A using these cells haspreviously not been described.

Adenoviruses identified from five clinical specimens based on directantigen testing (IFA) were detected in the H292 and cell culture mixes,while the PRMK cells missed two of the low titer specimens (i.e., therewere two false negatives). Thus, H292 and the mixed cultures were moresensitive than PRMK for detection of adenoviruses. While the A549 cellsmay provide slightly more positive cells, the 292 cells, mixed cellcultures, and A549 cells detected an equal number of positive specimens.

Parainfluenza viruses were also detected in the H292 and mixed cellcultures, while the PRMK cells missed one low titer specimen.

These results clearly show that the mixed cell cultures were equal insensitivity to the single cell (H292 and Mv1Lu) cultures. Thus, themixed cells provide savings in material, time, space, and labor, whileproviding the same level of sensitivity in the detection of respiratoryviruses as single cell cultures presently commonly used in diagnosticvirology laboratories.

Influenza B Specimens

In addition to the samples discussed above, various dilutions ofmultiple Influenza B strains obtained from the ATCC were tested in MDCK,Mv1Lu, and PRMK cells. The following table provides the results of theseexperiments. In this Table, “MD” refers to the “Maryland” strain, “HK”refers to the “Hong Kong” strain, “TW” refers to the “Taiwan” strain,and “MA” refers to the “Massachusetts” strain.

TABLE 3 Comparison of Influenza B Virus Detection From Prototype Virusesby MDCK, ML, and PRMK Cells Influenza B Virus Virus Cell Line StrainDilution MDCK Mv1Lu PRMK MD 10⁻⁴ + + + 10⁻⁵ + + + 10⁻⁶ − + − HK10⁻⁴ + + + 10⁻⁵ + + − 10⁻⁶ − − − TW 10⁻⁴ + + + 10⁻⁵ + + + 10⁻⁶ − − − MA10⁻⁴ + + + 10⁻⁵ + + + 10⁻⁶ + + +

These results-indicate that Mv1Lu, MDCK, and PRMK are comparable for thedetection of multiple Influenza B virus strains. Thus, these cell lineswere identified as good candidates for mixed cell cultures, as well assingle cell cultures for the identification of this virus.

EXAMPLE 3 Detection of CMV in Mixed Cell Cultures

In this Example, mixed cell cultures of Mv1Lu and NCI-H292 cells wereused to detect the presence of human cytomegalovirus (HCMV).

The Towne strain of HCMV (ATCC #VR977) was amplified in MRC-5 cells to atiter of greater than 10⁶/ml, and frozen at −85° C. in EMEM containing10% FBS. Serial dilutions of HCMV were prepared and inoculated intosingle monolayers of mink lung (Mv1Lu) cells, MRC-5 cells, and mixedcell monolayers of Mv1Lu and H292 cells. Each infected cell culturesystem was centrifuged for 60 minutes at 700×g, and then incubated for24 hours at 36° C. in 5% CO₂, in appropriate culture medium (e.g., EMEMcontaining 10% FBS). The culture medium was removed and the cells werefixed to the glass coverslip using a solution of 80% acetone in water. Asufficient amount of HCMV antibody solution (Chemicon) was added tocover each coverslip and incubated for 30 minutes at 35-37° C. Theantibody solution was removed, and the coverslip was rinsed with PBS. Asecond antibody solution consisting of FITC-labelled goat anti-mouseantiserum was added to cover each coverslip and incubated an additional30 minutes at 35-37° C. The specimens were then observed underepifluorescence at 100-400×magnification for positive (i.e.,CMV-infected), nuclear staining, fluorescent cells.

As described in previous Examples, the counterstain in the FITC-labelledgoat anti-mouse antibody solution contains Evans Blue, which stains thecells and appears red, when excited by fluorescent light. Fluorescent,apple green nuclear stain was observed in the Mv1Lu single cellmonolayer and in the mixed cell monolayers, but not in the H292 cells,as the Mv1Lu cells are susceptible to HCMV infection, while H292 cellsare not (or the H292 cells are significantly less sensitive). The MRC-5cells (i.e., the “gold standard” cells for detection of HCMV) performedabout the same as the mixed cell monolayer, as these cultures had asimilar number of infected cells as the cells in the mixed monolayer.

EXAMPLE 4 Detection of Enteroviruses in Mixed Cell Cultures

In this Example, mixed cell cultures were used to detect theenteroviruses, Coxsackie B virus and Echovirus. In these experiments, amixed cell monolayer of BGMK and NCI-H292 cells were used.

Confluent T-225 flasks of BGMK and H292 cells were prepared in EMEM withmM HEPES, 10% FBS, 2 mM L-glutamine, and 50 μg/ml gentamicin. The cellswere harvested by first rinsing in 30 ml HBSS without magnesium andcalcium, and were then dissociated from the flasks by a brief treatmentof 7 ml trypsin-EDTA solution (as described in Example 1). Then, anadditional 30 ml of culture medium (EMEM with HEPES, 10% FBS, 2 mML-glutanine, and 50 μg/ml gentamicin) was added to the suspension toproduce a cell suspension concentrate. The optical density at 500 nm wasdetermined for each suspension, using 3 ml of cells. Typically, the ODreading was 0.2/ml for both the BGMK and H292 cell suspensions.

Next, 3 ml of BGMK cell suspension and 8 ml of H292 cell suspension(both suspensions were at 0.2 OD units/ml) were then added to 29 ml ofthe culture medium (25 mM HEPES, 10% FBS, 2 mM L-glutamine, and 50 μg/mlgentamicin) to provide an acceptable working ratio of each cell type ina diluted mixed cell suspension. This ratio was intended to achieve aconfluent monolayer consisting of each cell type covering substantiallyequivalent surface area within 1-3 days post-planting of the dilutedmixed cells. Care was exercised to prepare a homogenous suspension ofdiluted mixed cells prior to dispensing 0.75 ml to each of 100 glassshell vials, each of which contained a sterile glass coverslip. Thevials were allowed to sit for 60 minutes post-planting at ambienttemperature to allow the cells to settle by gravity and produce a moreoptimum cell distribution. The vials were then were moved to anincubator for incubation at 36° C. for 1-3 days in 5% CO₂, at 95%relative humidity.

Stock virus suspensions and clinical specimens shown to containCoxsackie B virus or echovirus were used to infect BGMK/H292 cellmixtures, as well as single cell monolayers of BGMK, H292, MRC-5, andPRMK cells. For clinical samples, throat swab, nasopharyngeal swab,sputum, stool, and rectal swabs were collected from patients, placed inviral transport medium, and filtered through 0.45 μm filter to removepossible bacterial and fungal contaminants prior to inoculation of cellcultures. Cerebrospinal fluid (CSF) collected from patients was placedin viral transport medium, and used directly for inoculation of cells.For inoculation of shell vials, the media present in the vials wereremoved and fresh media added. Then, 0.2 ml of specimen was inoculatedinto each vial. The inoculated vials were centrifuged at 700×g for 45-60minutes at room temperature. Subsequently, the vials were incubated at37° C. for 1-3 days, and viral presence was detected usingimmunofluorescent staining.

For staining, the medium was removed from each vial and the cells werefixed on the coverslip with acetone. The coverslip was removed from eachvial, and stained with 25 μl primary antibody (mouse monoclonal antibodydirected against enteroviruses [Dako]), for 30 minutes at 37° C. Afterwashing with PBS, 25μl of the FITC-conjugated anti-mouse Ig (Dako) wasused as a secondary antibody for staining, and incubated at 37° C. for30 minutes. After another wash, the coverslips were mounted on slidesand observed under fluorescence. The presence of one or more specificfluorescent-stained cells on the coverslip was considered to be apositive. As described in previous Examples, the counterstain in theFITC-labelled goat anti-mouse antibody solution contains Evans Blue,which stains the cells, and appears red upon exposure to fluorescentlight. For Coxsackie B virus detection, fluorescent, apple green stainwas observed in many of the BGMK cells in the BGMK single cell monolayerand in the mixed cell monolayers primarily in the BGMK cells, but not inas many H292 cells, as BGMK cells are more susceptible to Coxsackie Bvirus infection. For some types of Coxsackie B virus isolates, H292cells are not as susceptible (or the H292 cells are significantly lesssusceptible). The “gold standard” cell line (i.e., PRMK cells) did notexhibit the same number of infected cells as the mixed cell monolayers.

For detection of echovirus, fluorescent, apple green stain was observedin many H292 cells in the H292 single cell monolayer and in the mixedcell monolayers, primarily in the H292 cells, but not in as many BGMKcells, as H292 cells are more susceptible to echovirus infection, whileBGMK cells are not as susceptible (or the BGMK cells are significantlyless sensitive). The “gold standard” line (i.e., MRC-5 cells) performed,but did not appear to have as many infected cells as the mixed cellmonolayers. In the case of the BGMK/H292 mixed cell monolayers infectedwith high titer samples of enteroviruses, cell-specific virus mediatedcytopathic effect (CPE) was evident (i.e., the CPE was observed in BGMKcells when Coxsackie B virus was present at high titer, and CPE wasobserved in H292 cells when echovirus was present at high titer).

EXAMPLE 5 Detection of herpes Simplex Virus and HCMV in Mixed CellCultures

In this Example, mixed cell cultures are used to detect herpes simplexvirus (HSV) and HCMV, using a mixed cell monolayer of geneticallyengineered baby hamster kidney (BHK) cells (e.g., ATCC #CCL-12072) andMv1Lu cells.

The BHK and Mv1Lu cells are grown in flasks, trypsinized, and mixed asdescribed in previous Examples, such that a suitable dilution of mixedcells is produced. These mixed cell dilutions are then used to inoculatesterile glass shell vials containing coverslips, as described above. Thecells are then centrifuged and inoculated with virus or clinicalsamples, incubated, and fixed, as described above.

HCMV is detected in the Mv1Lu cells, using antibody as described inExample 3 above, and HSV (HSV-1 and HSV-2) are identified using aβ-galactosidase staining kit (i.e., detecting the reporter gene inducedby the virus infecting the genetically engineered BHK cells).

EXAMPLE 6 Detection of Respiratory Viruses in Mixed Cell Cultures

In this Example, mixed cell cultures are used to detect a panel ofrespiratory viruses. In these experiments, three cell types are combinedso as to produce a mixed cell culture that is capable of detecting atleast three viruses.

First, A549, H292, and mink lung (e.g., Mv1Lu) cells cells are grown inflasks, trypsinized, and mixed as described in previous Examples, suchthat a suitable dilution of mixed cells is produced. In preferredembodiments, the cells are diluted such that the mixed cells in culturewill be in approximately the same proportions (i.e., 1:1:1). These mixedcell dilutions are then used to inoculate sterile glass shell vialscontaining coverslips, as described above. The cells are thencentrifuged and inoculated with virus or clinical samples, incubated,and fixed, as described above.

The viruses capable of infecting these cells are detected and identifiedusing the methods described in Example 2 above. In these mixed cellcultures, the 292 cells are used to detect the presence of parainfluenzaviruses and RSV, while the A549 cells are used to detect the presence ofadenoviruses, and the mink lung cells are used to detect the presence ofinfluenza viruses (e.g., Influenza A and B).

EXAMPLE 7 Detection of HSV and Chlamydia in Mixed Cell Cultures

In this Example, mixed cell cultures are provided which allow thedetection of two organisms commonly associated with sexually transmitteddiseases. In these experiments, mink lung cells (e.g., Mv1Lu) useful forthe detection of HSV are mixed with McCoy cells useful for the detectionof C. trachomatis.

First, McCoy cells and mink lung (e.g., Mv1Lu) cells cells are grown inflasks, trypsinized, and mixed as described in previous Examples, suchthat a suitable dilution of mixed cells is produced. In preferredembodiments, the cells are diluted such that the mixed cells in culturewill be in approximately the same proportions. These mixed celldilutions are then used to inoculate sterile glass shell vialscontaining coverslips, as described above. The cells are thencentrifuged and inoculated with samples (e.g., clinical samples),incubated, and fixed, as described above.

The organisms capable of infecting these cells (e.g., HSV infects themink lung cells, while C. trachomatis infects the McCoy cells) aredetected and identified using the methods described in Example 2 above.As with the other mixed cell culture systems, the presence of virusand/or C. trachomatis may be detected by other methods, such as theobservation of CPE, animal inoculation, etc. Thus, it is not intendedthat the mixed cell culture assay systems of this Example or any of thepreceding examples be limited to any particular method of microorganismdetection, identification, and/or quantitation.

From the above, it is clear that the present invention provides manyadvantages over presently used methods in diagnostic microbiology.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled indiagnostic microbiology and virology, cell culture, and/or relatedfields are intended to be within the scope of the following claims.

What is claimed is:
 1. A composition comprising cells suitable for thedetection of enterovirus, wherein said cells comprise buffalo greenmonkey kidney cell and a first cell type selected from humanmucoepidermoid cell and human lung carcinoma cell.
 2. The composition ofclaim 1, wherein said human mucoepidermoid cell is H292.
 3. Thecomposition of claim 1, wherein said human lung carcinoma cell is A549.4. The composition of claim 1, wherein said enterovirus is selected fromCoxsackie viruses, polioviruses, and echoviruses.